The CED4/Apaf1 family of proteins are intracellular, receptor-like molecules that coordinate the assembly of signaling complexes that regulate the activation of NF-kB, cytokine processing, and apoptosis. Human members of this family include: Apaf1, CARD4 (Nod1), Nod2 (CARD15), CARD 7 (DEFCAP/NAC/NALP1) and CARD12 (Ipaf/Clan) (Zou et al. (1997) Cell 90:405; Bertin et al. (1999) J. Biol. Chem. 275:41082; Inohara (1999) J. Biol Chem. 276:4812; Ogura et al. (2001) Nature 411:603; Bertin and DiStefano (2000) Cell Death Differ 7:1273; Hlaing et al. (2001) J. Biol Chem. 276:9230;, Chu et al. J. Biol. Chem. 276:9239; Geddes et al. (2001) Biochem. Biophys. Res. Commun. 284:77; Poyet (2001) J. Biol. Chem. 276:28309; Mactinon et al. (2001) Curr. Biol. 11:118; and Damiano et al. (2001) Genomics 75:77); PCT US 99/255544; PCT US 00/17691; and PCT US 00/29643. Each family member contains a CARD domain that mediates assembly with a downstream CARD-containing signaling partner, and a nucleotide-binding site that regulates activation of the signaling complex. In addition, each member contains a domain of either WD-40 repeats (Apaf1) or leucine-rich repeats (LRR; CARD4, CARD7, Nod2 and CARD12) that function as binding sites for upstream regulators. The NBS/LRR structure of CARD4, CARD7, Nod2 and CARD12 is strikingly similar to the plant NBS/LRR family of signaling proteins that induce gene expression and cell death in response to pathogen infection. However, plant NBS/LRR proteins contain either a leucine zipper motif or a Toll/interleukin-1 receptor homology region in place of a CARD domain. The regulation of NF-kB signaling by CARD4 and Nod2, and the activation of caspase-1 by CARD12 identifies these CED4/Apaf1 family members as important components of inflammatory signaling pathways. Consistent with this hypothesis, mutations within the Nod2 gene have been found to confer susceptibility to several chronic inflammatory disorders, including Crohn's disease and Blau syndrome (Hugot et al. (2001) Nature 411:537; Miceli-Richard et al. (2001) Nat. Genet. 29:19).
Nuclear factor-κB (NF-κB) is a transcription factor expressed in many cell types and which activates homologous or heterologous genes that have κB sites in their promoters. Molecules that regulate NF-κB activation play a critical role in both apoptosis and inflammation. Quiescent NF-κB resides in the cytoplasm as a heterodimer of proteins referred to as p50 and p65 and is complexed with the regulatory protein IκB. NF-κB binding to IκB causes to remain in the cytoplasm. At least two dozen stimuli that activate NF-κB are known (New England Journal of Medicine 336:1066, 1997) and they include cytokines, protein kinase C activators, oxidants, viruses, and immune system stimuli. NF-κB activating stimuli activate specific IκB kinases that phosphorylate IκB leading to its degradation. Once liberated from IκB, NF-κB translocates to the nucleus and activates genes with κB sites in their promoters. The proinflammatory cytokines TNF-α and IL-1 induce NF-κB activation by binding their cell-surface receptors and activating the NF-κB-inducing kinase, NIK, and NF-κB. NIK phosphorylates the IκB kinases α and β which phosphorylate IκB, leading to its degradation.
NF-κB and the NF-κB pathway has been implicated in mediating chronic inflammation in inflammatory diseases such as asthma, ulcerative colitis, rheumatoid arthritis (Epstein, New England Journal of Medicine 336:1066, 1997) and inhibiting NF-κB or NF-κB pathways may be an effective way of treating these diseases.
CARD7 and NBS1 are NBS/LRR proteins with N-terminal PYRIN-domains (Bertin and DiStefano, supra). The PYRIN domain is a protein-protein interaction module belonging to the death domain-fold superfamily which includes the CARD, death, and death effector domains (Bertin et al. (2000) J. Biol Chem. 275:41082; Pawlowski et al. (2001) Trends Biochem Sci. 26:85; Martinon et al. (2001) supra; Fairbrother et al. (2001) Prot. Sci. 10:1911. Proteins containing the PYRIN domain share homology with the N-terminal region of pyrin, a protein that functions to regulate inflammatory signaling in myeloid cells (Centola et al. (2000) Blood 95:3223). Mutations within the pyrin gene confer susceptibility to familial Mediterranean fever, a type of hereditary periodic inflammatory disease (The International FMF Consortium, 1997). In addition, the apoptosis proteins ASC and zebrafish caspase-13 each contain N-terminal PYRIN domains suggesting that PYRIN family members function in both inflammatory and apoptotic signaling (Masamuto et al. (1999) J. Biol. Chem. 274:33835; Inohara and Nunez, (2000) J. Biol. Chem. 276:2551.